A fundamental basis for life is the need and ability of an organism to generate energy. In humans, food is taken in and converted into chemical compounds such as adenosine triphospate (“ATP”) and nicotinamide adenine dinucleotide (“NAD”), which store the energy used by the cells of the body to perform the biological processes that sustain life. The metabolic pathways of the cells that convert the useful components of food (e.g., carbohydrates, fats and proteins) into usable energy are complex and may be affected by a variety of factors in ways that are not completely understood. Mammalian skin cells are no exception. Skin cells are known to include a variety of different kinds of cells that function together in a dynamic, complex relationship to maintain the health of the skin. For example, keratinocytes proliferate and differentiate to provide continuous skin turnover. Melanocytes are known to provide melanin synthesis for skin pigmentation. And fibroblasts are known for synthesizing the extracellular matrix and collagen, which helps maintain the skin's thickness and elasticity. Similarly, other cells found in or around the skin or other bodily organs, such as myocytes, stem cells, sebocytes, neurocytes, and adipocytes, all require energy derived from complex metabolic pathways, which can be undesirably impacted by a variety of different factors.
There is a growing awareness of the impact of various stressors on cellular bioenergetics and the impacts on cell aging, as well as other diseases (e.g., cancer, neurodegenerative diseases, diabetes, and cardiovascular disorders). One theory underlying some of these effects of altered metabolism in disease states is the Free Radical Theory of aging. Namely, that exposure of mammalian cells to reactive oxygen species (“ROS”) causes damage to cellular structures and organelles such as the mitochondria. ROS are highly reactive molecules that contain oxygen (e.g., oxygen ions and peroxides). ROS are formed naturally within cells as a natural byproduct of the normal metabolism of oxygen and play a role in cell signaling and homeostasis. However, when a cell is exposed to a stressor such as heat or UV radiation, ROS levels can increase, and in some instances dramatically.
As the damage caused by ROS accumulates over time, it causes more and more oxidative stress at the cellular level that ultimately may lead to tissue damage and/or organ dysfunction. One effect of oxidative stress on cells is a diminished capacity of cellular bioenergetics, which can lead to reduced levels of ATP and/or NAD. This may be particularly problematic for human skin because the oxidative stress on human skin cells may manifest as visible signs of aging. Further, environmental stressors such as ultraviolet radiation (“UV”) and pollutants (e.g., cigarette smoke, car exhaust, ozone) can lead to heightened levels of ROS production. Over time, this may result in noticeable changes in the skin's structure and morphology (e.g., “photodamage”) and, to a more extreme degree, skin carcinomas.
Human skin cells defend against ROS by using redox regulators such as glutathione and NAD as well as various enzymes that can neutralize ROS. However, these defenses can be overwhelmed by the elevated spike from stressor-induced ROS, leading to not just acute but also chronic alterations in energy homeostasis and metabolism efficiencies, causing overall cellular dysfunction. To complicate matters, the variety of cells types associated with human skin and the complexity of the metabolic pathways of these cells makes it difficult to identify suitable compounds to help combat the anti-aging effect associated with exposure to a particular oxidative stressor or combination of stressors. Certain oxidative stressors affect different types of cells and/or metabolic pathways differently. This makes it difficult to select a suitable skin care active or combination of actives (whose affect may also vary depending on type of cell or metabolic pathway) that combat the undesirable affects of a particular stressor or stressors. While identifying actives that prevent, reduce and/or reverse the effects of oxidative stress on a cell is desirable, it is even more desirable to identify combinations of actives that act together to provide an advantage over using a single active. This is sometimes referred to as synergy. Identifying skin-care actives that work alone to combat the effects of oxidative stress can be difficult, but quickly and/or efficiently identifying skin-care actives that work in synergy can be formidable. Thus, there is a long felt need for a method of identifying synergistic combinations of skin-care actives that combat the undesirable metabolic effects associated with various oxidative stressors, especially common environmental stressors.
As an initial step in finding synergistic combinations of skin-care actives, a method capable of detecting the changes in cellular metabolism caused by stressors and actives must be identified. Various methods are known for evaluating the energy making processes of cells. For example, Clark-type electrode probes are known for measuring oxygen consumption. The Clark electrode provides kinetic information (i.e., rates of response) but introduces artifact (i.e., some undesirable and/or extraneous factor that influences the results of a test) by its continuous consumption of oxygen, presenting a decreasing oxygen pressure to the cells or isolated mitochondria in the measurement chamber. Although oxygen consumption may provide an indication of mitochondrial function, it only measures one component of cellular bioenergetics and does not provide an assessment of other metabolic pathways that contribute to bioenergetic equilibrium, namely glycolysis.
Another conventional method for assessing cellular energy production is by measuring the amount of ATP in a cell. Luminescent ATP assay kits are commercially available for quantifying total energy metabolism. ATP assays are known to be relatively sensitive but they may not be an ideal metric of mitochondrial function as cells strive to maintain a particular ATP budget and will adjust metabolism accordingly. Thus, alterations in ATP levels are usually only detectable during pathophysiological changes. In addition, ATP assays are destructive (i.e., the cells are destroyed in order to measure the amount of ATP) and they lack kinetic information. Further, artifact has been reported from residual ATP present in dying or dead cells. And like the Clark-electrode assay, ATP cannot determine the relative contribution of different metabolic pathways to total ATP yield.
Still another convention method for assessing cell energetics is with commercially available MTT/XTT or alamarBlue™ kits. While these kits may provide a relatively simple way to assess cell health, they are not as sensitive as ATP assays. In addition, they have been reported to introduce error through cell toxicity, the very parameter they are supposed to be measuring. Further, both assays are destructive and do not provide kinetic information.
Accordingly, it would be desirable to provide a method of identifying and/or evaluating beneficial combinations of actives that reduce, prevent and/or reverse the undesirable oxidative stress on skin cells. It would also be desirable to provide a skin care composition that includes skin-care actives identified by the foregoing method. It would further be desirable to provide a method of treating skin damaged by the oxidative stress effects of a particular stressor.